Polypeptide based block copolymer and the process for the preparation thereof, and the polymer micelles using the same

ABSTRACT

The present disclosure relates to a polypeptide based block copolymer having biodegradability due to peptidase, and a process for the preparation thereof, and polymer micelles using the same. The block copolymer according to the present disclosure is a block copolymer of a polyethylene glycol-based compound having properties such that the solubility for water is different depending on the pH, but cannot form micelles due to a self-assembly phenomenon; and a polyglutamic acid-based compound formed using an aminolysis reaction of glutamic acid and tertiary amine in which the end of one alkyl group is substituted with NH2, or using an aminolysis reaction of glutamic acid and triamine. The block copolymer of the present disclosure has advantages in that the block polymer has both pH sensitivity and biodegradability due to peptidase in the body, and thereby a degradation rate related to a drug release cycle is controlled. Therefore, the block copolymer according to the present disclosure is effective in that the block copolymer can be used as a target-oriented drug delivery agent depending on the pH changes in the body, and as a drug delivery agent that can control the rate of degradation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. application Ser. No.14/022,356, filed on Sep. 10, 2013, now U.S. Pat. No. 9,180,199 issuedon Nov. 10, 2015, which claims the benefit under 35 USC 119(a) and365(b) of Korean Patent Application No. KR 10-2012-0100072, filed onSep. 10, 2012, in the Korean Intellectual Property Office.

TECHNICAL FIELD

The present disclosure relates to a polypeptide based block copolymerhaving biodegradability due to peptidase, and a process for thepreparation thereof, and polymer micelles using the same.

BACKGROUND ART

Micelles generally refer to a thermostable and uniform sphericalstructure formed by low-molecular weight materials havingamphiphilicity, that is, having a hydrophilic group and a hydrophobicgroup at the same time. When an insoluble drug is dissolved in andintroduced to a compound having the micelle structure, the drug ispresent inside the micelles, and these micelles can have highapplicability as a carrier for drug delivery since these micelles canperform target-oriented drug release in the body responding to changesin temperature and pH.

Korean Patent Application No. 10-2001-0035265 discloses a preparation ofmicelles using a polyethylene glycol and a biodegradable polymer. Thesematerials have advantages in that they all have bioaffinity due to theirbiodegradability, however, they have disadvantages in that drug deliveryin a target area is difficult since these materials are not sensitive tochanges in the body, for example, specific changes such as pH changes.

In addition, Korean Patent Application No. 10-2010-0112491 discloses apreparation of micelles using a block copolymer of a poly(β-aminoester)compound and a polyethylene glycol-based compound. These materials haveadvantages in that they are sensitive to specific changes such as pHchanges, however, they have disadvantages in that the control ofdegradation rate is impossible since degradation occurs due tohydrolysis after dimicellization.

Meanwhile, the pH environment in a body generally indicates pH 7.4 to7.2, however, it has been known that peripheral pH of abnormal cellssuch as cancer cells indicates pH 3.0 to 7.0, which is slightly acidicto strongly acidic. Recently, studies have been carried out in which adrug is released at pH 7.0 or lower in order to specifically deliver thedrug to cancer cells.

U.S. Pat. No. 5,955,509 having the tile of “pH dependent polymermicelles” discloses a method for preparing pH-sensitive polymer micellesin which a block copolymer of poly(vinyl N-heterocycle) andpoly(alkylene oxide) forms micelles at pH 6.0 or higher, and the blockcopolymer is collapsed between pH 2 and 6, and Japanese PatentApplication Laid-Open Publication No. 2002-179556 having the tile of“block copolymer-anti-cancer drug complex medicine” discloses a blockcopolymer of a hydrophilic polyethylene glycol-based compound and ahydrophobic polyamino acid-based compound forming micelles at a specificpH.

In view of the above, the inventors identified that, when a pH-sensitiveblock copolymer formed from a polyethylene glycol-based compound and apolyglutamic acid-based compound including a tertiary amine group isused, micelles are collapsed and thereby drugs can be released when pHis 7.0 or less, and micelles are formed and collapsed by the pHdifference of 0.2, therefore, the pH-sensitive block copolymer has bothpH sensitivity and biodegradability due to peptidase, and therebycontrol of the degradation rate relating to drug release cycle ispossible and the biotoxicity of the residues after degradation can bereduced, thereby completing the present disclosure.

DISCLOSURE Technical Problem

An object of the present disclosure is to provide a polypeptide basedblock copolymer having biodegradability due to peptidase, and a processfor the preparation thereof, and a polymer micelle-type diagnostic andtherapeutic composition including the block copolymer.

Technical Solution

In order to achieve the above object, the present disclosure provides apeptide based block copolymer represented by the following ChemicalFormula 1.

In the formula,

X is an integer of 10 to 200,

Y is an integer of 50 to 100,

R₁ is hydrogen or C₁₋₄ alkyl,

R₂s are each independently

and herein, 80% or more of y number of R₂s are i) the substituent A, orthe substituent C, the R₃ and R₄ are C₁₋₆ alkyl, the i is an integer of1 to n, the m_(i)s are each independently an integer of 1 to 6, and then is an integer of 1 to 6.

In addition, the present disclosure provides a polymer micelle-type drugcomposition that includes (a) the block copolymer; and (b) a molecularimaging marker for disease diagnosis, a contrast medium, or atherapeutic material including a bioactive substance for diseasetreatment, which can be included inside the block copolymer.

In addition, the present disclosure provides a method for preparing ablock copolymer, which includes the steps of preparing a compound of thefollowing Chemical Formula 2 by reacting glutamic acid, benzyl ester andtriphosgene (Step 1); preparing a compound of the following ChemicalFormula 4 by copolymerizing the compound of the following ChemicalFormula 2 with a compound of the following Chemical Formula 3 (Step 2);and reacting the compound of the following Chemical Formula 4 with acompound of the following Chemical Formula 5 or a compound of thefollowing Chemical Formula 6 (Step 3).

In the formulae, x, y, R₁, R₃, R₄, n, n₁ and n₂ are the same as thosedefined above.

Hereinafter, the present disclosure will be described in detail.

The polyglutamic acid-based compound block of the block copolymer of thepresent disclosure includes a tertiary amine group (—N═) that is ionizedat pH 7.0 or lower, and includes a to glutamic acid group that isdimicellized and degraded due to peptidase when the pH ranges from 6.5to 7.0. In addition, the polyglutamic acid-based compound block of theblock copolymer of the present disclosure includes

that is ionized at pH 7.0 or lower, and includes a glutamic acid groupthat is dimicellized and degraded due to peptidase when the pH rangesfrom 6.5 to 7.0.

R₂s of the Chemical Formula 1 of the present disclosure are eachindependently

and herein, 80% or more of y number of R₂s are the above substituent A.

In addition, in the present disclosure, it is preferable that 88% ormore of y number of R₂s of the Chemical Formula 1 be the substituent A.Furthermore, in the present disclosure, it is more preferable that 93%or more of y number of R₂s of the Chemical Formula 1 be the abovesubstituent A. In addition, in the present disclosure, it is even morepreferable that 96% or more of y number of R₂s of the Chemical Formula 1be the above substituent A.

Furthermore, R₂s of the Chemical Formula 1 of the present disclosure areeach independently

and herein, 80% or more of y number of R₂s are the substituent C.

The block copolymer of the present disclosure includes a tertiary aminegroup that is ionized at pH 7.0 or lower, and a glutamic acid group thatis degraded due to peptidase, therefore, the block copolymer issensitive to pH changes in the body, forms a micelle structure at aspecific pH range, is dimicellized and degraded due to peptidase whenthe pH ranges from 6.5 to 7.0 and thereby control of degradation rate ispossible.

Traditional poly(β-aminoester) compounds have an ester group in the mainchain and the chain is degraded due to hydrolysis, therefore,degradation products having a carboxylic acid group (—COOH) at the endare produced when the compound is degraded in the body. As a result,these degradation products, which have low acidity, can cause fataldamage to the tissues in the body. In contrast, in the presentdisclosure, a polymer is formed based on a polypeptide and degradationoccurs due to polypeptidase in the body, therefore the polymer hasbioaffinity since degradation products that have low acidity are notproduced.

In addition, when the polymer based on a polypeptide of the presentdisclosure is formed and the glutamic acid is used as a base, a blockcopolymer of the Chemical Formula 1 can be prepared by an aminolysisreaction between benzyl glutamic acid and tertiary amine in which theend of one alkyl group is effectively substituted with NH₂, and bycontrolling the reaction time of the aminolysis reaction, the ratio of

being converted to

can be controlled. Furthermore, when the conversion ratio is 80% ormore, pH-dependent properties are distinctively shown.

In addition, when a polyglutamic acid-based compound that includes atraditional tertiary amine group is used alone, pH-dependence is shown,however, micelles due to a self-assembly phenomenon cannot be formed.However, in the present disclosure, micelles due to a self-assemblyphenomenon can be formed by forming a block copolymer of a polyglutamicacid-based compound including a tertiary amine group and a hydrophilicpolyethylene glycol-based compound. Therefore, the block copolymerprepared according to the present disclosure can be used as a carrierfor target-oriented drug release as a diagnostic and therapeutic agentfor disease by forming a micelle structure capable of target release ata specific pH.

In addition, traditional drug delivery agents have problems in that thedrug accumulates in the body as a residue after the drug is delivered toa target area causing various side effects, and biodegradable polymersdesigned to avoid this problem also have problems in that the control ofdegradation rate is difficult since biodegradation occurs due tohydrolysis. Therefore, the copolymer is prepared using proteinconstituents such as glutamic acid as the monomer of the blockcopolymer, and there are advantages in that the dimicellized copolymeris biodegraded due to peptidase in the body and can be completelyeliminated from the body, and the degradation rate can be controlled.

The average molecular weight (Mn) of the copolymer of the presentdisclosure is not particularly limited, however, in the Chemical Formula1, it is preferable that x be an integer ranging from 10 to 200, and ybe an integer ranging from 50 to 100. In addition, it is more preferablethat the x be an integer of 10 to 100, and the y be an integer of 50 to100. If the value of the x is less than 10 and greater than 200, thecontrol of the molecular weight of the final block copolymer is not onlydifficult, but the formation of micelles using the block copolymer isnot simple. In addition, if the value of the y is less than 10, it isdifficult to form block copolymer micelles at a specific pH, and even ifthe micelles are formed, they tend to be dissolved in water andcollapsed. When the value of the y is greater than 200, the blockcopolymer may be precipitated without forming micelles at a specific pHsince hydrophilicity/hydrophobicity balance is broken.

In the Chemical Formula 1 of the present disclosure, R₁ is preferablymethyl. In addition, in the Chemical Formula 1 of the presentdisclosure, R₃ and R₄ are preferably isopropyl or n-butyl. Also, in theChemical Formula 1 of the present disclosure, n of

is preferably 2. In addition, in the Chemical Formula 1 of the presentdisclosure, n of

are preferably 2 or 5, and all m_(i)s thereof are preferably 2.

In the pH-sensitive micelles of the present disclosure, stable micellesare formed at a specific pH, for example, in the range of pH 7.2 to 7.8,which is the pH range of normal cells in the body, and the micellestructure is collapsed in the range of pH 6.5 to 7.0, which is the pHrange in which abnormal cells such as cancer cells are present.Therefore, the micelles can be used as a carrier for target-orienteddrug release, which can either diagnose targeting the cancer cells byreleasing the diagnostic agent included inside the micelles or treat thecancer cells by releasing the therapeutic agent included inside themicelles. In other words, micelles cannot be formed at a low pH (pH 7.0or lower) since the whole block copolymer becomes soluble due to theincrease of ionization of the tertiary amine present in the polyglutamicacid-based compound including the tertiary amine group, and in the rangeof pH 7.2 to 7.8, micelles due to self-assembly are formed sincehydrophobicity is shown due to the decrease of ionization of thetertiary amine.

The block copolymer of the present disclosure can be used in the fieldof gene transfer and drug delivery, and can also be applied to the usein which diagnosis and treatment are performed simultaneously bydelivering the substances for disease diagnosis and treatment to theabnormal cells.

In addition, in the present disclosure, target-oriented micellestargeting cancer cells are designed and applied in which micelles areformed in the range of pH 7.2 to 7.8, the same condition as that ofnormal body, and micelles are collapsed at pH 7 or lower, an abnormalcondition such as the presence of cancer cells. However, byappropriately changing the constituents of the block copolymer, themolar ratio thereof, molecular weight and/or functional groups withinthe block, target-oriented micelles targeting gene mutation or otherapplication fields besides cancer cells can be designed and applied.

As one of the constituents of the block copolymer that formspH-sensitive micelles according to the present disclosure, commonbiodegradable compounds having hydrophilicity known in the related art,for example, polyethylene glycol-based compounds, may be used. Inparticular, the polyethylene glycol-based compound preferably has amonofunctional group such as an amine group at the end so as to reactwith a polyglutamic acid-based compound, and one example includes acompound of the following Chemical Formula 3 in which the end of themolecule is substituted with NH₂.

In the formula, x is an integer of 10 to 200, and

R₁ is hydrogen or C₁₋₄ alkyl.

A method for preparing the pH-sensitive block copolymer according to thepresent disclosure includes the step of preparing a compound of thefollowing Chemical Formula 2 by reacting glutamic acid, benzyl ester andtriphosgene (Step 1); the step of preparing a compound of the followingChemical Formula 4 by copolymerizing the compound of the followingChemical Formula 2 with a compound of the following Chemical Formula 3(Step 2); and the step of reacting the compound of the followingChemical Formula 4 with a compound of the following Chemical Formula 5or a compound of the following Chemical Formula 6 (Step 3).

Each step of the preparation method will be described using one example.In the Step 1, in which the compound of the Chemical Formula 2 isprepared by reacting glutamic acid, benzyl ester and triphosgene,glutamic acid, benzyl ester and triphosgene are reacted first in thepresence of an anhydrous tetrahydrofuran solvent, and it is preferablethat the reaction be carried out under nitrogen atmosphere at 50° C.After the reaction has completed, the solution is introduced to hexane,recrystallized using 1:1 hexane/ethyl acetate as a recrystallizationsolvent, and the compound of the Chemical Formula 2 can be obtained.

In the Step 2, in which the compound of the Chemical Formula 4(PEG-b-PBLG) is prepared by copolymerizing the compound of the ChemicalFormula 2 with the compound of the Chemical Formula 3, the compound ofthe Chemical Formula 3 and benzyl-L-glutamic acid-N-carboxy anhydride(BLG-NCA) of the Chemical Formula 2 prepared in the Step 1 are reactedfirst by being dissolved in anhydrous chloroform in various molarratios. Preferably, the compound of the Chemical Formula 2 (BLG-NCA) andthe compound of the Chemical Formula 3 (PEG-NH2) are copolymerized in amolar ratio ranging from 10 to 1 to 50 to 1. It is preferable that theabove reaction be carried out under nitrogen atmosphere for 72 hours atroom temperature. After the reaction has completed, a copolymer of apolyglutamic acid-based compound block and a polyethylene glycol-basedblock, which has a molecular weight of Mn=10,000 to 20,000, is formed.

In the step of reacting the compound of the Chemical Formula 4 with thecompound of the Chemical Formula 5, part of y number of

of the Chemical Formula 4 are converted to

by controlling the time of reacting the compound of the Chemical Formula4 with the tertiary amine in which the end of one alkyl group of theChemical Formula 5 is substituted with NH₂.

In addition, in the step of reacting the compound of the ChemicalFormula 4 with the compound of the Chemical Formula 6, part of y numberof

of the Chemical Formula 4 are converted to

by controlling the time of reacting the compound of the Chemical Formula4 with the n-amine of the Chemical Formula 6.

In this case, the compound of the Chemical Formula 4 prepared in theStep 2 is dissolved in anhydrous N,N-dimethylformamide, and is reactedwith the compound of the Chemical Formula 5 or the compound of theChemical Formula 6. It is preferable that the reaction be carried out inan oil bath at 55° C. In addition, it is preferable that the reaction becarried out adding 2-hydroxypyridine (2-HP).

Furthermore, the reaction time in the Step 3 is preferably 36 hours to72 hours. By controlling the reaction time to be 36 hours to 72 hours,80% or more of y number of

can be converted to

In other words, an amine group is introduced depending on the reactiontime of the aminolysis reaction by converting

therefore, the conversion ratio increases as the reaction timeincreases. According to one example of the present disclosure, if thereaction time is less than 36 hours, the conversion ratio is less than80%, and the copolymer is not completely soluble in an aqueous solutionand does not have pH-sensitivity.

In the present disclosure, ¹H-NMR is used to measure the molecularweight of the block copolymer synthesized as above, and fluorescencespectrometer (FL) and dynamic light scattering (DLS) are used to measurethe changes in the micelle concentration depending on the changes in pHand the changes in micelle sizes, and the applicability as pH-sensitivemicelles was able to be verified through the analyses described above.

In a polymer micelle-type drug composition of the present disclosure,which includes (a) the block copolymer; and (b) a molecular imagingmarker for disease diagnosis, a contrast medium, or a therapeuticmaterial for disease treatment, which can be included inside the blockcopolymer, micelles are formed when the polymer micelle-type drugcomposition is injected into the body, and the micelles are collapsedwhen the polymer micelle-type drug composition reached to a regionhaving topically low pH such as cancer cells, and as a result,target-oriented drug delivery can be performed through the release ofthe molecular imaging marker for disease diagnosis, the contrast medium,or the therapeutic material for disease treatment, which are includedinside.

The molecular imaging marker, the contrast medium and the therapeuticmaterial that can be included in the polymer micelle-type blockcopolymer of the present disclosure can be used without particularlimitation Unlimited examples thereof include pyrene, RITC, FITC, ICG(Indocyanine Green), iron oxide, manganese oxide, or the like, as themolecular imaging marker, and anticancer drugs, antimicrobial agents,steroids, anti-inflammatory analgestic drugs, sex hormone drugs,immunosuppressive drugs, antiviral agents, anesthetic drugs, antiemeticdrugs, antihistamines, or the like, as the therapeutic drug. Inaddition, typical additives known in the related art such as dilutingagents, stabilizers, pH control agents, antioxidants, preserved agents,binding agents, disintegrating agents, or the like, may be included inaddition to the ingredients described above.

As the method for preparing the polymer micelles according to thepresent disclosure, methods such as stirring, heating, ultrasonic scan,a solvent evaporation method using an emulsification method, matrixformation, a dialysis method using an organic solvent can be used eitheralone or in combination.

The diameter of the prepared polymer micelles is not particularlylimited, however, the range of 10 to 200 nm is preferable. In addition,the polymer micelle drug composition can be used as a pharmaceuticalpreparation in the form of oral agents or non-oral agents, and may beprepared as intravenous injections, intramuscular injections orhypodermic injections.

Advantageous Effects

The block copolymer according to the present disclosure has advantagesin that a degradation rate is controlled since the block copolymer notonly has pH sensitivity but has biodegradability due to peptidase in thebody. In addition, the block copolymer has bioaffinity since theoccurrence of degradation products having a carboxylic acid group(—COOH) at the end due to the chain degradation by hydrolysis can beprevented. Furthermore, the block copolymer can be changed variously byvarying the amine compound used in the block copolymer preparation.Therefore, the block copolymer according to the present disclosure iseffective in that the block copolymer can be used as a target-orienteddrug delivery agent depending on the pH changes in the body, and as adrug delivery agent having bioaffinity, which can control the rate ofdegradation.

DESCRIPTION OF DRAWINGS

FIG. 1 shows an NMR of benzyl-L-glutamic acid-N-carboxy anhydride(BLG-NCA) prepared in Preparation Example 1.

FIG. 2 shows an NMR of poly(ethyleneglycol)-block-poly(benzyl-L-glutamic acid) (PEG-b-PBLG) prepared inPreparation Example 2.

FIG. 3 shows an NMR of poly(ethyleneglycol)-block-poly(2-(dibutylamino)ethyl-L-glutamic acid) (PEG-b-PN4LG)prepared in Example 4.

FIG. 4 is a graph that shows the ratio of the benzyl group ofpoly(ethylene glycol)-block-poly(benzyl-L-glutamic acid) (PEG-b-PBLG)described in the above Examples 1 to 4 and Comparative Examples 1 and 2being converted to a (dibutylamino)ethylamine group as the reaction timeprogresses.

FIG. 5 is a graph that shows a pKb value determined by titrimetry usinga NaOH aqueous solution of a polypeptide based block copolymer.

FIG. 6 shows the changes in the sizes of micelles and the changes in theintensity of dynamic light scattering (DLS) due to the pH changes ofpoly(ethylene glycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid)(PEG-b-PN4LG) prepared in Example 1 (36 h), Example 2 (48 h) and Example4 (72 h).

FIG. 7 shows the result of fluorescence analysis (FL) of the micellesdue to the pH changes of poly(ethyleneglycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid) (PEG-b-PN4LG)prepared in Example 1 (36 h), Example 2 (48 h) and Example 4 (72 h), andthe intensity of fluorescence analysis (FL) depending on the wavelength(nm) of poly(ethyleneglycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid) (PEG-b-PN4LG)prepared in Example 2 (48 h).

FIG. 8 shows the measurement results of critical micelle concentration(CMC) of poly(ethyleneglycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid) (PEG-b-PN4LG)prepared in Example 2 (48 h).

FIG. 9 shows the result of the gel retardation experiment.

FIG. 10 shows the result of GFP silencing.

FIG. 11 shows the result of MIT assay.

MODE FOR DISCLOSURE

Hereinafter, the present disclosure will be described in more detailwith reference to examples. However, these examples are for illustrativepurposes only and the scope of the present disclosure is not limited tothese examples.

Preparation Example 1: Preparation of Poly(EthyleneGlycol)-Block-Poly(Benzyl-L-Glutamic Acid) Copolymer

10 g of L-glutamic acid-benzyl ester and 6.255 g of triphosgene weremixed with 100 mL of anhydrous tetrahydrofuran. The reaction was carriedout under nitrogen atmosphere at 50° C. After 2 hours and the reactionhas completed, the above solution was introduced to hexane, and themixture was recrystallized twice using 1:1 hexane/ethyl acetate as arecrystallization solvent, resulting in benzyl-L-glutamic acid-N-carboxyanhydride (BLG-NCA). The yield was 73%. The ¹H NMR (500 MHz, CDCl₃)measurement result of the BLG-NCA obtained above is shown in FIG. 1.

The benzyl-L-glutamic acid-N-carboxy anhydride (BLG-NCA) prepared aboveand polyethylene glycol of

were dissolved in anhydrous chloroform in a molar ratio of 20 to 1. Thereaction was carried out under nitrogen atmosphere for 72 hours at roomtemperature. After the reaction has completed, poly(ethyleneglycol)-block-poly(benzyl-L-glutamic acid) (PEG-b-PBLG), a copolymer ofa polyglutamic acid block and a polyethylene glycol block, which has amolecular weight of Mn=11,570, was formed. The reactant that has notbeen reacted was precipitated using a mixture of excess ethanol/ethylether, and then filtered.

Preparation Example 2: Preparation of Poly(EthyleneGlycol)-Block-Poly(Benzyl-L-Glutamic Acid) Copolymer

The benzyl-L-glutamic acid-N-carboxy anhydride (BLG-NCA) prepared in thesame manner as in Example 1 and polyethylene glycol of

was dissolved in anhydrous chloroform in a molar ratio of 30 to 1. Thereaction was carried out under nitrogen atmosphere for 72 hours at roomtemperature. After the reaction has completed, poly(ethyleneglycol)-block-poly(benzyl-L-glutamic acid) (PEG-b-PBLG), a copolymer ofa polyglutamic acid block and a polyethylene glycol block, which has amolecular weight of Mn=13,541, was formed. The reactant that has notbeen reacted was precipitated using a mixture of excess ethanol/ethylether, and then filtered. The ¹H NMR (500 MHz, CDCl₃) measurement resultof the copolymer obtained above is shown in FIG. 2.

Preparation Example 3: Preparation of Poly(EthyleneGlycol)-Block-Poly(Benzyl-L-Glutamic Acid) Copolymer

The benzyl-L-glutamic acid-N-carboxy anhydride (BLG-NCA) prepared in thesame manner as in Example 1 and polyethylene glycol of

was dissolved in anhydrous chloroform in a molar ratio of 40 to 1. Thereaction was carried out under nitrogen atmosphere for 72 hours at roomtemperature. After the reaction has completed, poly(ethyleneglycol)-block-poly(benzyl-L-glutamic acid) (PEG-b-PBLG), a copolymer ofa polyglutamic acid block and a polyethylene glycol block, which has amolecular weight of Mn=17,045, was formed. The reactant that has notbeen reacted was precipitated using a mixture of excess ethanol/ethylether, and then filtered.

Example 1: Preparation of Poly(EthyleneGlycol)-Block-Poly(2-(Dibutylamino)-Ethyl-L-Glutamic Acid BlockCopolymer (PEG-b-PN4LG)

Poly(ethylene glycol)-block-poly(benzyl-L-glutamic acid) (PEG-b-PBLG)(10 g) prepared in the above Preparation Example 2 was dissolved inanhydrous N,N-dimethylformamide (10 mL/1 g). Then, the mixture wasintroduced in an oil bath, and the temperature was raised to 55° C.After that, 2-(dibutylamino)ethylamine (100 g) and 2-hydroxypyridine(13.82 g) were added. The reaction was carried out for 36 hours, and afinal product, poly(ethyleneglycol)-block-poly(2-(dibutylamino)ethyl-L-glutamic acid) (PEG-b-PN4LG)in which 80% of the benzyl group was converted to the dibutylaminogroup, was obtained after being precipitated using ether and thenfiltered. The final product was dried under vacuum condition for 48hours. A block copolymer in which the number average molecular weight ofthe whole block copolymer was 12,823, was obtained, and the yield was70% or more.

Example 2: Preparation of Poly(EthyleneGlycol)-Block-Poly(2-(Dibutylamino)-Ethyl-L-Glutamic Acid BlockCopolymer (PEG-b-PN4LG)

A block copolymer in which the number average molecular weight of thewhole block copolymer was 13,942 and the conversion ratio was 88% wasobtained by the same reaction as in Example 1 except that the reactiontime of 2-(dibutylamino)ethylamine and 2-hydroxypyridine addition was 48hours. The yield of the final product was 70% or more.

Example 3: Preparation of Poly(EthyleneGlycol)-Block-Poly(2-(Dibutylamino)-Ethyl-L-Glutamic Acid BlockCopolymer (PEG-b-PN4LG)

A block copolymer in which the number average molecular weight of thewhole block copolymer was 12,340 and the conversion ratio was 93% wasobtained by the same reaction as in Example 1 except that the reactiontime of 2-(dibutylamino)ethylamine and 2-hydroxypyridine addition was 60hours. The yield of the final product was 70% or more.

Example 4: Preparation of Poly(EthyleneGlycol)-Block-Poly(2-(Dibutylamino)-Ethyl-L-Glutamic Acid BlockCopolymer (PEG-b-PN4LG)

A block copolymer in which the number average molecular weight of thewhole block copolymer was 12,011 and the conversion ratio was 96% wasobtained by the same reaction as in Example 1 except that the reactiontime of 2-(dibutylamino)ethylamine and 2-hydroxypyridine addition was 72hours. The yield of the final product was 70% or more. The ¹H NMR (500MHz, CDCl₃) measurement result of the copolymer obtained above is shownin FIG. 3.

Example 5: Preparation of Poly(EthyleneGlycol)-Block-Poly(2-(Diisopropylamino)-Ethyl-L-Glutamic Acid BlockCopolymer (PEG-b-PN3LG)

Poly(ethylene glycol)-block-poly(benzyl-L-glutamic acid) (PEG-b-PBLG)(10 g) prepared in the above Preparation Example 2 was dissolved inanhydrous N,N-dimethylformamide (10 mL/1 g). Then, the mixture wasintroduced in an oil bath, and the temperature was raised to 55° C.After that, 2-(diisopropylamino)ethylamine (80 g) and 2-hydroxypyridine(13.82 g) were added. The reaction was carried out for 72 hours, and afinal product, poly(ethyleneglycol)-block-poly(2-(diisopropylamino)ethyl-L-glutamic acid)(PEG-b-PN3LG) in which 95% of the benzyl group was converted to thediisopropylamino group, was obtained after being precipitated usingether and then filtered. The final product was dried under vacuumcondition for 48 hours. A block copolymer in which the number averagemolecular weight of the whole block copolymer was 12,959, was obtained,and the yield was 70% or more.

Example 6: Preparation of Poly(EthyleneGlycol)-Block-Poly[(2-Aminoethyl)-2-Aminoethyl]-L-Glutamic Acid BlockCopolymer (PEG-b-PN2LG)

Poly(ethylene glycol)-block-poly(benzyl-L-glutamic acid) (5k-PEG-b-PBLG)(10 g) prepared in the above Preparation Example 2 was dissolved inanhydrous N,N-dimethylformamide (10 mL/1 g). Then, the mixture wasintroduced in an oil bath, and the temperature was raised to 55° C.After that, diethylenetriamine (60 g) and 2-hydroxypyridine (13.82 g)were added. The reaction was carried out for 72 hours, and a finalproduct, poly(ethyleneglycol)-block-poly[(2-aminoethyl)-2-aminoethyl]-L-glutamic acid)(PEG-b-PN2LG) in which 95% of the benzyl group was converted to the(ethylamino)₂ group, was obtained after being precipitated using etherand then filtered. The final product was dried under vacuum conditionfor 48 hours. A block copolymer in which the number average molecularweight of the whole block copolymer was 14,000, was obtained, and theyield was 70% or more.

Example 7: Preparation of Poly(EthyleneGlycol)-Block-Poly[N—(N—(N—(N—(N-(2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl]-L-GlutamicAcid Block Copolymer (5k-PEG-b-PN5LG)

Poly(ethylene glycol)-block-poly(benzyl-L-glutamic acid) (5k-PEG-b-PBLG)(5 g) prepared in the above Preparation Example 2 was dissolved inanhydrous N,N-dimethylformamide (10 mL/1 g). Then, the mixture wasintroduced in an oil bath, and the temperature was raised to 55° C.After that, pentaethylenehexaamine (67 g) and 2-hydroxypyridine (6.91 g)were added. The reaction was carried out for 72 hours, and a finalproduct, poly(ethyleneglycol)-block-poly[N—(N—(N—(N—(N-(2-aminoethyl)-2-aminoethyl)-2-aminoethyl)-2-aminoethyl)-2-aminoethyl]-L-glutamicacid block copolymer (5k-PEG-b-PN5LG) in which 95% of the benzyl groupwas converted to the (ethylamino)₅ group, was obtained after beingprecipitated using ether and then filtered. The final product was driedunder vacuum condition for 48 hours. A block copolymer in which thenumber average molecular weight of the whole block copolymer was 18,700,was obtained, and the yield was 70% or more.

Example 8: Preparation of Poly(EthyleneGlycol)-Block-Poly[N—(N—(N—(N—(N-(2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl]-L-GlutamicAcid Block Copolymer (3.4k-PEG-b-PN5LG)

Poly(ethylene glycol)-block-poly(benzyl-L-glutamic acid)(3.4k-PEG-b-PBLG) (5 g) prepared in the above Preparation Example 2 wasdissolved in anhydrous N,N-dimethylformamide (10 mL/1 g). Then, themixture was introduced in an oil bath, and the temperature was raised to55° C. After that, pentaethylenehexaamine (67 g) and 2-hydroxypyridine(6.91 g) were added. The reaction was carried out for 72 hours, and afinal product, poly(ethyleneglycol)-block-poly[N—(N—(N—(N—(N-(2-aminoethyl)-2-aminoethyl)-2-aminoethyl)-2-aminoethyl)-2-aminoethyl]-L-glutamicacid block copolymer (3.4k-PEG-b-PN5LG) in which 95% of the benzyl groupwas converted to the (ethylamino)₅ group, was obtained after beingprecipitated using ether and then filtered. The final product was driedunder vacuum condition for 48 hours. A block copolymer in which thenumber average molecular weight of the whole block copolymer was 17,000,was obtained, and the yield was 70% or more.

Example 9: Preparation of Poly(EthyleneGlycol)-Block-Poly[N—(N—(N—(N—(N-(2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl)-2-Aminoethyl]-L-GlutamicAcid Block Copolymer (2k-PEG-b-PN5LG)

Poly(ethylene glycol)-block-poly(benzyl-L-glutamic acid) (2k-PEG-b-PBLG)(5 g) prepared in the above Preparation Example 2 was dissolved inanhydrous N,N-dimethylformamide (10 mL/1 g). Then, the mixture wasintroduced in an oil bath, and the temperature was raised to 55° C.After that, pentaethylenehexaamine (67 g) and 2-hydroxypyridine (6.91 g)were added. The reaction was carried out for 72 hours, and a finalproduct, poly(ethyleneglycol)-block-poly[N—(N—(N—(N—(N-(2-aminoethyl)-2-aminoethyl)-2-aminoethyl)-2-aminoethyl)-2-aminoethyl]-L-glutamicacid block copolymer (2k-PEG-b-PN5LG) in which 95% of the benzyl groupwas converted to the (ethylamino)₅ group, was obtained after beingprecipitated using ether and then filtered. The final product was driedunder vacuum condition for 48 hours. A block copolymer in which thenumber average molecular weight of the whole block copolymer was 15,700,was obtained, and the yield was 70% or more.

Comparative Example 1: Preparation of Poly(EthyleneGlycol)-Block-Poly(2-(Dibutylamino)-Ethyl-L-Glutamic Acid BlockCopolymer (PEG-b-PN4LG)

A block copolymer in which the number average molecular weight of thewhole block copolymer was 11,115 and the conversion ratio was 40% wasobtained by the same reaction as in Example 1 except that the reactiontime of 2-(dibutylamino)ethylamine and 2-hydroxypyridine addition was 12hours. The yield of the final product was 70% or more.

Comparative Example 2: Preparation of Poly(EthyleneGlycol)-Block-Poly(2-(Dibutylamino)-Ethyl-L-Glutamic Acid BlockCopolymer (PEG-b-PN4LG)

A block copolymer in which the number average molecular weight of thewhole block copolymer was 11,216 and the conversion ratio was 63% wasobtained by the same reaction as in Example 1 except that the reactiontime of 2-(dibutylamino)ethylamine and 2-hydroxypyridine addition was 24hours. The yield of the final product was 70% or more.

Test Example 1. Evaluation of Conversion Ratio by Reaction Time

The poly(ethylene glycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamicacid) copolymer (PEG-b-PNLG) prepared in Examples 1 to 6 was sampled bythe reaction time, and the conversion ratio was evaluated using ¹H-NMR(FIG. 4). In other words, the conversion ratio was evaluated using¹H-NMR by measuring the increase of a specific peak area of PNLG havinga tertiary amine group. As shown in the following Table 1, it wasidentified that the ratio of conversion to the tertiary amine groupincreased as the reaction time increased.

TABLE 1 Conversion Polymer Type Reaction Time Ratio PreparationPEG-b-PBLG 72 h — Example 1 Preparation PEG-b-PBLG 72 h — Example 2Preparation PEG-b-PBLG 72 h — Example 3 Comparative PEG-b-PN4LG 12 h 40%Example 1 Comparative PEG-b-PN4LG 24 h 63% Example 2 Example 1PEG-b-PN4LG 36 h 80% Example 2 PEG-b-PN4LG 48 h 88% Example 3PEG-b-PN4LG 60 h 93% Example 4 PEG-b-PN4LG 72 h 96% Example 5PEG-b-PN3LG 72 h 95% Example 6 PEG-b-PN2LG 72 h 95%

Test Example 2. Measurement of Changes in Micelles Due to Changes in pHTest Example 2-1. Measurement of pKb Value of pH-Sensitive Copolymer

A pKb value was measured by titrimetry using a NaOH aqueous solutionutilizing the block copolymer (PEG-b-PNLG) prepared in the Examples 1 to5, which has different molecular weights depending on the reaction time(FIG. 5). As shown in FIG. 5, it was verified that the more the reactiontime increased, that is, the more the PNLG having a tertiary amine groupwas produced, the acid-based inflection point was changed rapidly, andthe pKb value more or less increased. The measured pKb value is shown inthe following Table 3.

Test Example 2-2. Measurement of Changes in Micelle Sizes and DynamicLight Scattering (DLS) Intensities Due to pH Changes of pH-SensitiveCopolymer

The changes in micelle sizes and intensities of poly(ethyleneglycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid) (PEG-b-PN4LG)prepared in Examples 1 to 6 were measured at pH 7.8, pH 7.4, pH 7.2, pH7.0, pH 6.8 and pH 6.6 using a dynamic light scattering method (DLS)(FIG. 6).

As the result of the test, it was seen that micelles having certainsizes are present at pH 7.0 or higher, however, it was identified that,at a pH of lower than 7.0, PN4LG was completely ionized and micelleswere not formed at all.

As a result, it was verified that the block copolymer of the presentdisclosure, which was formed by the copolymerization of hydrophobicPN4LG and a hydrophilic PEG compound, can form or collapse polymermicelles through a reversible self-assembly phenomenon due to pH changesand the amphiphilicity present within the copolymer.

In addition, it was identified that the copolymers prepared inPreparation Examples 1 to 3 and the copolymers of which reaction timewas adjusted to 12 hours to 24 hours were insoluble or not completelysoluble in an aqueous solution (Table 2).

TABLE 2 Solubility in Polymer Type Reaction Time Aqueous SolutionPreparation PEG-b-PBLG 72 h Insoluble Example 1 Preparation PEG-b-PBLG72 h Insoluble Example 2 Preparation PEG-b-PBLG 72 h Insoluble Example 3Comparative PEG-b-PN4LG 12h Insoluble Example 1 Comparative PEG-b-PN4LG24 h Not Completely Example 2 Soluble Example 1 PEG-b-PN4LG 36 h pHdependent Example 2 PEG-b-PN4LG 48 h pH dependent Example 3 PEG-b-PN4LG60 h pH dependent Example 4 PEG-b-PN4LG 72 h pH dependent Example 5PEG-b-PN3LG 72 h pH dependent Example 6 PEG-b-PN2LG 72 h pH dependent

Test Example 2-3. Measurement of Changes in Fluorescence Analysis (FL)of pH-Sensitive Copolymer

The changes in the fluorescence analysis (FL) of the micelles due to thepH changes of poly(ethyleneglycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid) (PEG-b-PN2LG)prepared in Example 1, Example 2 and Example 4 were measured (FIG. 7).Pyrene, a hydrophobic light emitting material, was used since thebehavioral changes of the micelles cannot be directly determined using afluorescence spectrometer.

A buffer solution of pH 6 containing 10⁻⁶M of pyrene was prepared, andthe pH of the solution was raised to pH 8.0 after each copolymerprepared in Examples 1 to 6 was dissolved to have the concentration of 1mg/ml. After that, 5M hydrochloric solution was added dropwise so thatthe pH was changed to the range of 5.5 to 8.0, and the changes in theemitted energy due to the concentration changes of the micelles weremeasured using a fluorescence spectrometer.

Test Example 2-4. Measurement of Critical Micelle Concentration (CMC) ofpH-Sensitive Copolymer

The critical micelle concentration (CMC) of poly(ethyleneglycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid) (PEG-b-PN4LG)prepared in Examples 1 to 4 was measured at pH 6.8, 6.9, 7.0 and 7.1(FIG. 8).

As the result of the test, in the block copolymer of the presentdisclosure, it was verified that stable micelles were formed at pH 7.4,and no micelles were formed at pH 7.0 (FIG. 8). This means that thecopolymer cannot form micelles at a low pH (pH 7.0 or lower) since thewhole PNLG becomes soluble due to the increase of ionization of thetertiary amine present in PNLG, and at pH 7.4, micelles due toself-assembly are formed since hydrophobicity is shown due to thedecrease of ionization of PNLG. The measured critical micelleconcentration values are shown in the following Table 3.

Test Example 2-5. Size Measurement of pH-Sensitive Copolymer

The size of poly(ethyleneglycol)-block-poly(2-(dibutylamino)-ethyl-L-glutamic acid) (PEG-b-PNLG)prepared in Examples 1 to 4 was measured at pH 7.4 using a dynamic lightscattering (DLS) method. The measured diameter values of the micellesare shown in the following Table 3.

TABLE 3 CMC Size Polymer Type Mn pKb (mg/mL) (pH 7.4) PreparationPEG-b-PBLG 11,570 — — — Example 1 Preparation PEG-b-PBLG 13,541 — — —Example 2 Preparation PEG-b-PBLG 17,045 — — — Example 3 ComparativePEG-b-PN4LG 11,115 — — — Example 1 Comparative PEG-b-PN4LG 11,216 — — —Example 2 Example 1 PEG-b-PN4LG 12,823 6.32 0.020 46.0 nm Example 2PEG-b-PN4LG 13,942 6.55 0.019 45.2 nm Example 3 PEG-b-PN4LG 12,340 6.650.019 42.0 nm Example 4 PEG-b-PN4LG 12,011 6.69 0.017 40.0 nm Example 5PEG-b-PN3LG 12,959 8.30 — — Example 6 PEG-b-PN2LG 14,000

Test Example 3. Evaluation of Gel Retardation

To confirm the initial ratio of complex formation between cationicpolymers prepared according to the present invention and anionic geneticmaterials, the following analysis was performed.

With variation of each mass ratio of the prepared polymer and siRNA, acomplex was formed, and subsequently the formation of the complex wasconfirmed via electrophoresis using 1 wt % agarose gel as a matrix. As aresult, it was confirmed that, when the polymer/siRNA ratio was 1, thecomplex was not properly formed due to siRNA separation, whereas whenthe ratio was 3 or higher, the complex was properly formed (FIG. 9).

Test Example 4. Evaluation of GFP Silencing

To confirm transfer efficiency of the mPEG-b-PNLG/siRNA complex formed,the following GFP silencing was performed.

One hundred thousand cells were cultured in each well of a 12-well plateand were then treated with the complex prepared by the same manner inTest Example 3. After 24 hours of treatment, cells in each well werescraped together using a lysis buffer solution, and a protein analysiswas performed. The type of cells used in the experiment is A549, whichexpresses Green Fluorescent Protein (GFP), and the siRNA used therein isone that inhibits GFP expression. Accordingly, as a smaller amount ofexpressed GFP was used, a larger amount of siRNA was transferred insidethe cells.

As for the results of GFP silencing, it was confirmed that as the massratio of polymer/siRNA increased, the level of GFP expression ratiosignificantly decreased throughout the experiment. Further, when theresults were compared with bPEI 25k, which is commonly used for thetransfer of siRNA into the cells, it was confirmed that all polymersexhibited a significantly lower GFP expression ratio once the mass ratioof polymer/siRNA reached 10 or higher. Such an observation implies thatall polymers prepared exhibit superior efficiency compared to bPEI (FIG.10).

Test Example 5. MTT Assay

To confirm the toxicity of the cationic polymers prepared according tothe present invention, the following experiment was performed.

Ten thousand cells were cultured for 24 hours in each well of a 96-wellplate and were then treated with polymer/microRNA complex with varyingratios. After 24 hours of treatment, the cells were treated with an MITsolution to confirm the number of living cells. Herein, as more cellssurvived, yellow MIT tetrazolium was reduced to purple MIT formazan, andthus it was possible to determine the extent of cell survival.

As a result, it was confirmed that, as the polymer/microRNA complexratio of PEG having a molecular weight of 2k increased, the ratio ofliving cells was about 60%, indicating some toxicity. In contrast, inthe case of PEG having molecular weights of 3.4k and 5k, the ratio ofliving cells was over 80%, indicating no toxicity, regardless of thecomplex ratio (FIG. 11).

The invention claimed is:
 1. A peptide based block copolymer representedby the following Chemical Formula 1:

wherein, X is an integer of 10 to 200; Y is an integer of 50 to 100; R₁is hydrogen or C₁₋₄ alkyl; R₂s are each independently

wherein, 80% or more of y number of R₂s are the substituent C; and the iis an integer of 1 to n, the m_(i)s are each independently an integer of1 to 6, and the n is an integer of 1 to
 6. 2. The block copolymer ofclaim 1, wherein 88% or more of y number of R₂s are the substituent C.3. The block copolymer of claim 1, wherein 93% or more of y number ofR₂s are the substituent C.
 4. The block copolymer of claim 1, wherein96% or more of y number of R₂s are the substituent C.
 5. The blockcopolymer of claim 1, wherein R₁ is methyl in the Chemical Formula
 1. 6.The block copolymer of claim 1, wherein n is 2 to 5 in the ChemicalFormula
 1. 7. The block copolymer of claim 1, wherein all m_(i)s are 2in the Chemical Formula
 1. 8. A polymer micelle-type drug compositioncomprising: (a) the block copolymer of claim 1; and (b) a molecularimaging marker, a contrast medium or a therapeutic material, which areincluded inside the block copolymer.
 9. The polymer micelle-type drugcomposition of claim 8, wherein 88% or more of y number of R₂s are thesubstituent C.
 10. The polymer micelle-type drug composition of claim 8,wherein 93% or more of y number of R₂s are the substituent C.
 11. Thepolymer micelle-type drug composition of claim 8, wherein 96% or more ofy number of R₂s are the substituent C.
 12. The polymer micelle-type drugcomposition of claim 8, wherein R₁ is methyl in the Chemical Formula 1.13. The polymer micelle-type drug composition of claim 8, wherein n is 2to 5 in the Chemical Formula
 1. 14. The polymer micelle-type drugcomposition of claim 8, wherein all m_(i)s are 2 in the ChemicalFormula
 1. 15. A method for preparing a peptide based block copolymerrepresented by the following Chemical Formula 1:

X is an integer of 10 to 200; Y is an integer of 50 to 100; R₁ ishydrogen or C₁₋₄ alkyl; R₂s are each independently

wherein, 80% or more of y number of R₂s are the substituent C; and the iis an integer of 1 to n, the m_(i)s are each independently an integer of1 to 6, and the n is an integer of 1 to 6, comprising the steps of:preparing a compound of the following Chemical Formula 2 by reactingglutamic acid, benzyl ester and triphosgene (Step 1); preparing acompound of the following Chemical Formula 4 by copolymerizing thecompound of the following Chemical Formula 2 with a compound of thefollowing Chemical Formula 3 (Step 2); and reacting the compound of thefollowing Chemical Formula 4 with a compound of the following ChemicalFormula 5 or a compound of the following Chemical Formula 6 (Step 3),

wherein, the reaction of Step 3 comprises breaking a C—O bond of anester moiety of Chemical Formula 4 and forming a C—N bond of an amidmoiety, and the time of reacting the compound of the Chemical Formula 4with Chemical Formula 5 or Chemical Formula 6 is controlled such thatpart of y number of

of Chemical Formula 4 are converted to substituent C such that at least80% or more of y number of R₂s are the substituent C.
 16. The method forpreparing the block copolymer of claim 15, wherein the reaction timeranges from 36 hours to 72 hours in the Step 3.